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Ribonuclease S
RNase S is RNase A treated with subtilisin, which cleaves a single peptide bond. Consequently, Ribonuclease S consists of two fragments of bovine Ribonuclease A in a peptide-protein complex: S peptide (amino acids 1-20) and S protein (amino acids 21-124). RNase S was the third enzyme and fourth protein ever cystallized and three-dimensional structure determined. As such, biological work on RNase S helped scientists determine the first 3-dimensional structure of a protein-nucleic acid complex. Additionally, RNase S provided one of the first demonstrations of a crystalline enzyme acting as an active catalyst. Investigations of RNase S have also led to many technological advancements, including substrate leash amplification, fusion protein systems, and protein ubiquitination. RNase S has been studied to reveal mechanisms of protein folding by coupling folding and association. Studies of RNase S has lead to greater interest in the investigation of RNase A and its role in biological systems, and has also lead to a greater knowledge of the correlation between protein folding and enzyme activity.



RNase A and RNase S have very similar structures except for a few key areas, one being the cleavage site (residues 16-23). Decreased order in these amino acids is seen in RNase S. The cleavage site on RNase A is located between residues Ala 20 and Ser 21. Upon cleavage, the fragments S peptide and S protein are created, giving RNase S its unique structure. S Peptide consists of residues 1-20, and is largely responsible for proper folding. S protein is comprised of residues 21-124. Additionally, RNase A and RNase S have many conserved structural components, as they are essentially the same protein. Glutamine 60 is highly conserved structurally between RNase S and RNase A, as well as in many different species, demonstrating its importance. Glu 60 is also interesting, because it is the only residue in an unfavorable position (Φ= -100, ψ= -130) as defined in the Ramachandran plot.



The active site of RNase S consists of residues His 12, Lys 41, Val 43, Asn 44, Thr 45, His 119, Phe 120, Asp 121, and Ser 123. His 12 is the only residue in the active site that is located on the S peptide fragment. Lys 41 and His 119 are also crucial to the functioning of RNase S. Interestingly, both catalytic Histidines perform the same function, even though they are located on different fragments. Residues 1, 15-20, 21-23, and 124 could be removed without serious consequence to the structure or activity of RNase S. Residue 124 could also be removed from RNase A without compromising the structure.



Hydrogen bonding and hydrophobic interactions play a major role in the structure of RNase S. There are 84 water molecules present, with 8 of these specifically connecting S peptide and S protein. Some of these water molecules are also conserved throughout all RNase derivatives. Hydrophobic interactions between residues Phe 8, Met 13, His 12, Ala 4 are essential in holding the S Peptide in place. In addition to these residues, Asp 14 is also important in peptide-protein binding. In the picture, it appears that the protein and peptide are not connected; this is because the bond cleavage between residues 20 and 21 of RNase A has already occurred. Additionally RNase S has a rigid hydrophobic core; one-third of the surface of the core is made up of the S peptide. The surrounding loops have more flexibility.

References

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Other resources:

Wikipedia article on RNase A: http://en.wikipedia.org/wiki/Rnase_A

RNase S on PDB: http://www.rcsb.org/pdb/explore/explore.do?structureId=1RNU

RNase A on PDB: http://www.rcsb.org/pdb/explore/explore.do?structureId=7RSA

Ribonuclease on Proteopedia: http://proteopedia.org/wiki/index.php/Ribonuclease